A nonmitochondrial (cyanide-insensitive) O2 utilizing reaction in neoplastic cells injured by macrophages has been studied. The first objective was to completely purify L1210 leukemia cells following co-culture with cytotoxic macrophages. This was required to prove that the nonmitochondrial O2 consumption is a reaction occurring in target neoplastic cells and not contaminating macrophages. A fluorescent antibody method was used to quantitate the number of remaining macrophages in L1210-cell suspensions removed from co-culture vessels. The hybridoma, MAC-1, monoclonal antibody did not react with L1210 cells but did bind to the surface of murine macrophages. Purification of L1210 cells by passage over plastic to remove adherent cells did not remove all MAC-1 positive cells. Consequently, other methods of purification are required. Thus far, additional methods have failed to yield macrophage-free L1210 cell suspensions. Step or continuous Percoll centrifugal density gradients yield mixed L1210-cell macrophage collections. Increasing phagocyte density by exposure to carbonyl iron proved toxic for L1210 cells. Passage of L1210 cells through Sephadex G-10 or nylon wool columns to remove contaminating macrophages resulted in very low recovery of leukemia cells (less than 10% of input number). An alternative to purification has been attempted. The cyanide-insensitive O2 consumption is completely blocked by killing the cells with detergent, for example, digitonin, triton, or lubrol. Thus, if contaminating macrophages can be selectively killed, their potential contribution to this reaction can be assessed. Resident murine macrophages overlaid with MAC-1 antibody plus guinea pig complement are rapidly killed. All cells become trypan blue positive following a 30 min incubation. However, when cytotoxic-mouse macrophages were tested, no killing occurred. This is despite the fact that these macrophages bind MAC-1 antibody as determined using the fluorescence microscope. (MB)